Tailoring and characterization of chitosan with well-defined pattern of acetylation

Chitosan is a linear binary copolymer composed of glucosamine and N-acetylglucosamine units, exhibiting excellent bioactivities and broad application potential. The structural architecture of chitosan is defined by three fundamental parameters: degree of polymerization (DP), fraction of acetylation (FA), and pattern of acetylation (PA). However, due to long-standing constraints in preparation and analytical methodologies, current research predominantly concentrates on DP and FA, while largely overlooking the structural parameter of PA. This review systematically summarizes the production and characterization methods of chitosan with well-defined PA, and highlights that PA is an important factor regulating the structure-function relationship of chitosan. Importantly, PA can significantly modify its physicochemical properties and bioactivities independently of DP and FA. These advances have primarily relied on the increasing availability of well-characterized recombinant chitin deacetylases (CDAs), as well as continuously optimized techniques such as chromatographic separation and mass spectrometry. Together, these developments have enabled systematic research on chitosan with well-defined sequences and predictable properties.